Biomedical Engineering Reference
In-Depth Information
mannosidase IC enzyme appeared to digest these oligosaccharides more readily
than the α-1,2-mannosidases IB enzyme even though the α-1,2-mannosidase IB had
a higher specifi c activity towards the synthetic substrate Man-α-1,2-Man-OCH
3
.
This difference may be related to certain structural differences which limit the
access of these intermediates within the binding pocket to the catalytic region of
the enzyme. Manipulation of the expression of these components of the N-glycan
processing pathway will be an important step in glyco-engineering this important
fungal expression strain.
INTRODUCTION
Filamentous fungi, particularly those of the genera
Aspergillus
and
Trichoderma
, are widely used for the heterologous expression of proteins
as they are capable of producing up to 20-30 grams of protein per litre of
culture (Kalsner et al. 1995, Archer and Peberdy 1997, Gouka et al. 1997,
Withers et al. 1998, Maras et al. 1999). Filamentous fungi possess many
other signifi cant advantages as protein expression systems, including
ease of genetic manipulation, a high level of adaptability allowing
exploitation of a wide range of environmental conditions and nutrient
sources, well developed and diverse promoter systems and a structural
morphology amenable to high cell culture density and simple biomass
fi ltration (Lubertozzi and Keasling 2009). An important consideration
in the production of non-fungal enzymes in fungal hosts is the fi delity
of post-translational processing events, such as protein glycosylation
(reviewed in Hamilton and Gerngross 2007, De Pourcq et al. 2010). The
post-translational addition of non-authentic N-glycans to proteins used
as therapeutic agents can result in reduced activity or stability, increased
serum clearance and can sometimes result in an adverse immune response
(Jenkins et al. 1996, Jenkins 2007, Jacobs and Callewaert 2009). To utilize
fi lamentous fungi for the production of such specialized glycoproteins,
it is preferable to produce glycoproteins which carry carbohydrate
structures as similar to the natural product as possible. Manipulation
of the N-glycosylation pathway to produce glycoproteins with 'correct'
N-glycan structures would dramatically increase the utility of these
expression systems for the production of therapeutic protein drugs.
Engineering of glycan specifi c protein expression has been accomplished
in several diverse systems, including: the yeasts
Pichia pastoris
(Choi et al.
2003, Bobrowicz et al. 2004, Vervecken et al. 2004, Wildt and Gerngross
2005, Hamilton and Gerngross 2007),
Ogataea minuta
, (Kuroda et al. 2006),
Saccharomyces cerevisiae
(Chiba and Jigami 2007, Chiba and Akeboshi 2009),
Hansenula polymorpha
(Oh et al. 2008) and
Yarrowia lipolytica
(Song et al.
2007); the fi lamentous fungi
Trichoderma reesei
(Maras et al. 1997b, Maras
et al. 1999),
Aspergillus spp.
(Kainz et al. 2008); insect cell lines (Hollister
